Name: __________________________________ Date: ______________ Class: 1 2 3 4 5 6 7
The Case so far . . .
You have identified the strain of bacteria that infected the residents as ___________________.
You have identified the ink using ____________________________ and determined that the contaminated salad
bag came from _________________________________.
Gel Electrophoresis of Ink from SALAD Packaging
Testable Question: Can gel electrophoresis be used to confirm the paper chromatography results from the
contaminated salad packaging?
Background Information: Agarose gel electrophoresis allows you to separate molecules according to size
and electrical charge. What is agarose? It is a polymer that is made entirely of sugar molecules. It is
produced by a kind of seaweed and is used to give thicker consistency to foods such as ice cream. We use
agarose because a solution of agarose and water forms a gel when it cools to room temperature. It is sort
of like jello except that it does not get soft when it gets warm.
Molecules like ink dyes can move through an agarose gel, but just like in paper chromatography the larger
they are, the slower they move. To understand the process, think about a backyard that is full of trees. If
you watch, you can see that small birds fly through the branches of the trees almost as if the trees were
not there. What about a larger bird like a hawk or an owl? They have a harder time flying through the gaps.
Therefore, they cannot fly as fast as the smaller birds. In the same way, small molecules can move quickly
among the agarose branches in the gel, but larger molecules move more slowly because they have to find
larger gaps to pass through.
What is electrophoresis? Electrophoresis is a process that uses electricity to pull molecules from one place
to another. If you look at the gel box, you will see that it has two bare wires called electrodes. One is
connected to a red wire and has a positive charge, and the other is connected to a black wire and has a
negative charge. Many, but not all, dyes have a negative charge so they are attracted by the positive
charge and move through the gel towards the positive electrode. Therefore, we are going to use agarose gel
electrophoresis to pull dye molecules through an agarose gel and separate them according to size and
positive charge.
Like the paper chromatography technique you have already used, this technique will allow you to separate
the mixtures of the inks. You will compare the ink gel patterns used on the salad bags of four salad
processing plants to the ink gel pattern on the bag that contains the contaminated salad. Then you will use
your data to conclude which bag (and therefore which processing plant) matches and is responsible for
distributing the contaminated salad.
Mission review:
1.
What is agarose made from? ____________________________________________________
2. What is gel electrophoresis?
__________________________________________________________________________
__________________________________________________________________________
Name: __________________________________ Date: ______________ Class: 1 2 3 4 5 6 7
3. How is agarose gel electrophoresis used to separate mixtures of molecules?
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
4. How will agarose gel electrophoresis be used to confirm the results of the paper chromatography
and prove which local farm produced the contaminated salad?
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
Materials:
Salad Bag A Ink from Schneider’s Salads
Salad Bag B Ink from Spivey’s Salads
Salad Bag C Ink from Endler’s Endless Greens
Salad Bag D Ink from Grandusky’s Gardens
Contaminated Salad Packaging (x)
Agarose gel
TBE Buffer
Gel apparatus
Power supply
Pipettes and pipette
Gloves and goggles
Procedures (Check off each step as you complete it):
Safety Precautions

Follow your teacher’s directions in regarding micropipette handling.

The power supply produces a voltage that is high enough to cause severe electrical shock if handled
improperly. Ensure you know the procedures regarding the use of this unit, making electrical connections, and
that you are directly supervised by the teacher.

Do NOT plug power supply into wall receptacle until the safety cover is positioned on the cell and all other
electrical connections are properly made.

Make sure that all electrical equipment is dry.

Do not come in personal contact with or allow metal or any conductive material to come in contact with
reservoir buffer or the electrophoretic cell while power supply is on.

The power supply may continue to produce some voltage even when the power has been turned off. When
disconnecting the gel box be sure that the leads do not touch each other, come in contact with the buffer
solution, or otherwise create any hazardous electrical condition.
Name: __________________________________ Date: ______________ Class: 1 2 3 4 5 6 7
Black
electrode (-)
Loading and running the samples
____1.
On the gel, load 10 uL of each ink sample into a corresponding lane.
Keep track of which sample goes into which lane on the data sheet. Use a
new tip for each sample and be careful not to puncture the bottom of
the well. Your teacher will demonstrate.
Casting
tray tab
____2.
Clean up any spilled buffer or any other liquid surrounding the gel
box thoroughly.
____3.
Make sure the power supply is unplugged and switched off
before proceeding.
____4.
Carefully place the lid on the gel box (first making sure it is clean
and dry) and connect the terminals correctly to the power supply.
____5.
Have your teacher look over your connections before plugging in
your power supply.
____6.
Plug in the power supply, switch it on and adjust the voltage to 100
volts. You will know that current is circulating through your unit if you see
tiny bubbles forming on the electrodes in the gel box.
Fig. 1
Red electrode
(+)
____7.
Let your gel run for 10 - 12 minutes. Keep checking your gel to make sure the ink molecules
do not run off the end of the gel.
____8.
When the fastest moving molecules are approximately 1 to 2 cm from the end of the gel,
turn off the power supply. Disconnect the cords from the power supply and unplug the power
supply.
____9.
Ask your teacher to remove the gel tray from the gel box carefully – the gel is slippery and
it can slide off and break. Place the tray on a flat surface and record your results on your data
sheet. It will help if your gel tray is on a light background.
____10.
Follow your teacher’s instructions for clean up. Micropipette tips may be discarded in the
trash. Gel boxes and trays must be rinsed and air dried. Wash your hands thoroughly and clean
the area where you were working.
STUDENT ACTIVITY Data and Analysis
1. Why is the comb placed in the center for this electrophoresis experiment?
Name: __________________________________ Date: ______________ Class: 1 2 3 4 5 6 7
2. The diagram below represents your gel.
a.
As you load the ink samples into your gel, record which sample was placed into which well
using the table below.
b.
After you have run the gel, use colored pencils or markers to draw in the results of your ink
separation.
Black electrode (-)
A
B
C
D
E
F
Red electrode (+)
Lane Number
Factory Name/Source of Sample
A
________________________________
B
________________________________
C
________________________________
D
________________________________
E
________________________________
F
________________________________
Name: __________________________________ Date: ______________ Class: 1 2 3 4 5 6 7
3.
Which ink mixture likely contained the smallest molecule? ___________ Use evidence
from your gel to explain how we know this.
4.
Which ink mixture contained the largest molecule? ___________ Use evidence
from your gel to explain how we know this.
5.
List the inks that have molecules with a negative charge. ________________
_____________ ________________ __________________ _______________ Use evidence from
your gel to explain how we know this.
6.
List the inks that have molecules with a positive charge. ___________
_____________ ______________ ________________ ______________Use evidence
from your gel to explain how we know this.
7.
Which salad bag matches the contaminated salad bag sample? ____________Use
evidence from your gel to explain how we know this.